Prior-art electrochemical sensors typically consist of an electrochemical cell with two, sometimes three electrodes. The first electrode is responsive to a chemical species in the test solution and is called the indicator electrode. The second electrode called the reference electrode is typically non-responsive to changes in the composition of the test solution. In polarography a third, current-injecting counter electrode is sometimes used.
As is appreciated by those in the art, the performance of an electrochemical sensor as part of a chemistry analyzer for quantitative measurement of chemicals in aqueous solutions is determined by its dose-response curve. For a linear sensor this can be uniquely determined by two coefficients: a slope and an intercept. For a dose-response curve that is non-linear, three or more coefficients may be required. As is also known in the art, a sensor's coefficients vary over time if it is used more than once. The coefficients also vary from sensor to sensor because no two sensors can be manufactured identically. Therefore, a calibration is generally required to uniquely determine a sensor's dose-response curve. In an automated chemistry analyzer the calibration is provided by fluidic elements (calibration fluids, pumps, valves, conduits etc.) contained within the analyzer. If a sensor is deployed as a reusable device it is often the case that the chemistry analyzer's calibration fluidics provides for at least two calibration points and a wash solution. This is because slope and intercept of the dose-response curve can change through repeated uses. For a unit-use device no calibration would be required if the slope and intercept were sufficiently reproducible from sensor to sensor during manufacture and storage. A single calibrator would be required if either one of the coefficients was reproducible, the other not, and two calibrators if neither coefficient was reproducible (more calibrators could be required for devices with non-linear dose-response curves).
Often the goal of a manufacturer of chemistry analyzers is to produce sensors sufficiently cheaply so that they can be deployed as unit-use devices, thus eliminating or simplifying the chemistry analyzer's often very complex fluidics required for the washing and calibrating of multiple-use sensors. To this end, manufacturers have investigated planar technologies for low cost sensor manufacture. Such technologies also purport to provide appropriate control of the materials of construction and manufacturing processes to achieve device-to-device reproducibility in high volume production.
Sensors made by planar technology have included both thick-film and thin-film micro-fabrication technologies. Thick film processed devices such as plastic diagnostic strips are disclosed in U.S. Pat. No. 5,727,548 for example. Devices made by planar technology also include thick film processed planar substrates as in hybrid circuit or printed circuit manufacture. U.S. Pat. Nos. 4,133,735, 4,225,410 for example disclose devices with electrodes made by thick film fabrication processes such as plating, screen-printing, dispensing and the like.
Micro-fabrication technology with its proven superior dimensional control also has been used to make devices for unit-use applications. Micro-fabrication technology employs wafer-level processes such as photolithography on silicon wafers. U.S. Pat. Nos. 4,062,750 4,933,048 and 5,063,081 disclose devices containing electrodes made by thin-film micro-fabrication processes on silicon substrates.
Regardless of which of the above variants of planar technology is being used, planar devices of the prior art have been complex to manufacture and are therefore still expensive.
To better appreciate the complexity of prior-art planar sensors, consider their typical components of construction. A planar electrochemical sensor of the prior art is a device consisting of one or more metal conductor elements on a planar insulating substrate. One region of the metal conductor element is provided for connection to an external measuring circuit. A planar electrode is formed in another region of the metal conductor element. The planar electrode of such a prior-art electrochemical sensor consists typically of one or more additional metal layers (or other electrical conductors such as graphite) and insoluble metal salt over-layers over-coating the metal conductor element. Planar electrodes are typically then coated with several additional functional layers as outlined below.
The planar electrode of the planar sensor is typically coated by an integral electrolyte medium. This integral electrolyte may be a liquid aqueous solution or, more commonly, a solid hydrophilic layer such as a gel material that acts like an aqueous electrolyte. In use of the planar sensor, the planar electrode region and its integral electrolyte over-layer is immersed in an aqueous solution to be tested. Chemical species from the test solution permeate into the integral electrolyte layer, dissolve and often react with other reagents contained within the integral electrolyte layer. Components of the integral electrolyte layer undergo electrochemical reaction at the electrode surface generating a current or a voltage. When the measured current or voltage of the sensor is selectively proportional to the concentration of a species in the test solution that is transported from the test solution into the sensor there is the basis for an indicator electrode for that species. If the voltage is independent of test solution composition there is the basis for a reference electrode. In prior-art electrochemical sensors it is generally required that chemical reagents within the integral electrolyte layer be at constant concentrations during the time of the measurement.
It is generally required that chemicals contained within the test solution that are deleterious to the sensor reactions be rejected from the integral electrolyte layer. As is known in the art such contaminants may affect chemical reactions within the integral electrolyte layer, or they may themselves be electro-active and cause a voltage or current that interferes with the measured voltage or current due to the species being analyzed. Retention of reagent chemical and rejection of contaminants is achieved by interposing one or more materials between the integral electrolyte and the test solution. Transport of the sensed species from the test solution into the integral electrolyte layer takes place by selective diffusion through the interposed materials. In many cases of prior-art planar sensors it is also necessary to interpose an additional semi-permeable layer between the electrode and the integral electrolyte layer. The purpose of this electrode-modifying layer is to allow transport of the chemicals of the sensor reaction while rejecting electroactive interferents or species that poison the electrode.
In summary, as described above, planar electrochemical sensors of the prior art including the prior-art reference electrodes, enzyme electrodes and gas sensing electrodes generally consist of numerous elements. The resulting devices are complex and costly to manufacture. To further illustrate their complexity, the devices of the prior art in each of the above categories addressed by the current invention are described in more detail in the following sections.
Potentiometric Salt-Bridge Reference Electrode Prior Art
Salt-bridge reference electrodes of the prior art consists of an electrode, usually silver with a silver chloride over-layer which is contacted by an integral reservoir of a concentrated aqueous solution of a salt with equi-mobile ions, typically potassium chloride. The electrolyte reservoir contacts the test solution through a constrained-flow liquid junction, which is typically a micro-porous element. The integral aqueous electrolyte reservoir and the junction together comprise a salt bridge. An ideal salt-bridge reference electrode of this design has an essentially constant electrode potential and essentially zero response slope for the duration of its use. As is known in the art of reference electrodes, the total electrode potential is the sum of the potential difference between the electrode and integral salt-bridge electrolyte and the liquid-junction potential difference which is between the salt-bridge electrolyte and the test solution. The constant electrode potential of such prior-art reference electrodes is achieved firstly because the potential determining chloride concentration of the salt-bridge electrolyte at the silver-silver chloride electrode surface remains essentially fixed for the duration of use. This is achieved both because the rate of chloride efflux from the reservoir into the test solution is sufficiently small because of the constrained-flow junction and because the electrolyte reservoir is sufficiently large. Secondly, the response slope of such salt-bridge reference electrode is also small when the liquid junction potential difference is small as is the case when the salt-bridge electrolyte contains a concentrated salt with anions and cations of nearly equal mobility, such as with the use of a concentrated potassium chloride electrolyte.
Planar potentiometric salt-bridge reference electrodes of the prior art have used the same approach as the classical salt-bridge reference electrode described above. U.S. Pat. No. 4,592,824 describes a planar silver-silver chloride electrode on a planar silicon substrate, and a silicon cover-plate including a micro-fabricated cavity and porous region. The cavity including the porous junction becomes the integral salt-bridge reservoir when it is filled with concentrated potassium chloride before use. The porous silicon element forms the region of the constrained-flow liquid junction that contacts the test solution. Similarly, U.S. Pat. No. 4,682,602 describes a planar silver-silver chloride electrode and a cover layer defining a cavity over the electrode. The cavity, when filled with electrolyte, becomes the integral salt-bridge reservoir. There is a small aperture providing a flow-constraining liquid junction contact to a test solution. U.S. Pat. No. 5,385,659 describes a planar silver-silver chloride with a micro-fabricated, elongated cavity in a cover plate. When the elongated cavity is filled with electrolyte it becomes the integral salt bridge reservoir. The flow of electrolyte out of the salt-bridge is constrained because the cavity is elongated and its opening is small. These and other prior-art planar reference electrodes with integral electrolyte cavities are relatively complex and costly assemblies. They must be filled with concentrated salt-bridge electrolyte before use, or, if filled in the factory, they must be stored wet. Consequently, they are impractical for unit-use applications.
U.S. Pat. No. 4,342,964 describes a fluidic cassette for blood measurement containing a dry-stored silver-silver chloride electrode without an integral salt-bridge electrolyte over-layer and a spaced apart indicator electrode. In use, a calibrator solution is introduced over the pair of electrodes serving to calibrate the indicator electrode prior to its subsequent exposure to the test solution. The calibrator solution also fills an empty cavity region of the cassette over the silver-silver chloride electrode and remains there to form a liquid junction with the test solution when it is subsequently introduced into the cassette. Thus, this patent teaches how to automatically fill a reference electrode's salt-bridge reservoir without significantly adding to the complexity of the reference electrode itself, because the device already requires a calibrator solution and the patent teaches that the calibrator solution can be the same as the salt-bridge filling solution. However there is added fluidic complexity and cost, and the significant limitation on this invention is that there is no single composition of the calibrator solution that is satisfactory both to accurately calibrate the indicator electrode and provide for a low-response liquid junction. For acceptable performance in blood it is known in the art that the salt-bridge electrolyte should have a potassium chloride concentration of about 1M or even larger for the liquid junction potential component of the reference electrode to be acceptably small and constant. Known calibrator solutions for blood do not provide this concentration
Janata in Solid State Chemical Sensors, Janata J. and Huber R. J. (eds.), Academic Press Inc., Orlando 1985, pp 101-103, describe an ion-sensitive field effect reference electrode with an integral salt-bridge reservoir formed by a hydrophilic gel layer coating the electrode. Sinsabaugh et al. in Proceedings, Symposium on Electrochemical Sensors for Biomedical Applications, Vol. 86-14, Conan, K. N. L. (ed.), The Electrochemical Society, Pennington, N.J. 1986, pp 66-73, describe a planar reference electrode consisting of a silver-silver chloride electrode over-coated by an integral salt-bridge reservoir formed by a latex membrane. In this device there are in total three coating steps onto the conductor element and its support. The Janata and Sinsabaugh devices were intended for multi-use sensor applications utilizing a calibrator solution. In a typical measurement the reference electrode, with its salt-bridge reservoir over-layer, and a spaced-apart indicator electrode are first immersed in a calibrator solution. The integral reservoir equilibrates to the concentration of the calibrator solution. When the electrode-pair is then immersed in a test solution the indicator electrode responds rapidly but, because of its integral constrained-flow reservoir, the potential difference between the silver-silver chloride and the salt-bridge electrolyte over-layer responds slowly. If the reservoir thickness is sufficient (several hundred micrometers) the response is slow enough to constitute a constant potential over the time that the indicator electrode responds (approximately 10 s). During multiple uses the composition of the salt-bridge gradually approaches the concentration of the calibrator and test solutions in which it is immersed. These reference electrodes in multi-use application are once again limited in utility for accurate blood measurements because the liquid junction component of the reference electrode potential is not sufficiently small or constant because the salt-bridge reservoir concentration is too low. Both these papers are silent on the use of their salt-bridge reservoirs as dry-reagent formulations in unit-use reference electrodes. Both papers are silent on the incorporation of redox chemicals into the salt-bridge reservoirs and the use of such in reference electrodes constructed with salt-bridges coating metals. The Sinsabaugh paper is also silent on the water vapor transport properties of their latex membrane formulation.
Because of the complexity of manufacture of reference electrodes containing integral fluid reservoirs and because of the difficulty of their storage and preparation for use, a dry-reagent reference electrode is highly desirable for unit-use applications. An integral dry-reagent salt-bridge reservoir that contains only dry salts must first acquire water so that the salt-bridge reservoir can ‘wet up’ to its operational concentration. In all of the above-mentioned prior-art devices the transport of species through the salt-bridge reservoir and from the salt bridge to the contacting solution is through an electrolyte phase. Water influx for wet-up of the prior-art devices dry reagent devices is through the same path as potassium chloride efflux. Thus, in a device featuring a constrained flow salt-bridge design with a sizeable reservoir that is required to maintain constancy of chloride concentration at the silver-silver chloride surface, the time for water uptake also will be large. Also, the potassium chloride of the salt bridge electrolyte will escape from the reservoir into the solution while the reservoir is acquiring water from the solution for its wet-up. Therefore, reference electrodes with dry reagent reservoirs according to the above prior art have not been successfully deployed in unit-use applications.
The above wet-up problem was addressed in U.S. Pat. No. 4,933,048, which describes a dry-reagent salt-bridge reference electrode made by planar micro-fabrication. In this device there is a first insulating layer on a planar substrate that supports a conductor for connection to a measuring circuit. A second insulating layer covers the conductor except in a region that defines the electrode opening. There are films of silver, then silver chloride formed over the conductor in the electrode region. A solid hydrophilic material containing potassium chloride is formed over the silver chloride. This layer constitutes the integral salt-bridge reservoir. In this device, the salt-bridge reservoir extends well beyond the silver-silver chloride electrode edge and is further coated by a hydrophobic water vapor-permeable over-layer, except for a region of the salt bridge that is far removed from the silver-silver chloride where the salt-bridge contacts the test fluid defining the liquid junction. This unit-use salt-bridge reference electrode was designed to rapidly wet-up during use from its dry storage state, and to essentially retain a constantly high concentration of potassium chloride in the integral salt-bridge reservoir for a period after full wet-up and through the time of the measurement. These desired properties are obtained in the device of the '048 patent by providing a short diffusion path for rapid water influx into the integral reservoir through the water vapor-permeable over-layer and a long diffusion path for the potassium chloride in the salt-bridge along the length of the integral reservoir. In use, the water necessary for the proper function of the salt bridge is rapidly incorporated into the initially dry potassium chloride layer within a few seconds by diffusion through the gas permeable over-layer. The concentration of the internal salt-bridge electrolyte rapidly reaches a steady state value after a wet-up period of a few seconds which is maintained for a period sufficient to perform the potentiometric measurement. However, this device is complex to manufacture, consisting of five layers on top of the conductor element and its insulating support.
U.S. Pat. No. 4,431,508 describes a graphite reference electrode with a hydrophilic coating containing a redox couple manufactured with non-planar conventional technology.
In summary, planar reference electrodes of the prior art consist of a silver-silver chloride electrode contacting an integral salt-bridge electrolyte reservoir consisting of concentrated potassium chloride. These devices are either manufactured with water already incorporated into the salt-bridge reservoir, or, they are dry-reagent devices with a gas permeable coating that facilitates water transport into the salt bridge. The salt bridge makes connection to the test solution through a small, flow-constraining orifice or other flow limiting physical constriction fabricated on the device in planar technology. The connection of the salt bridge to the test solution is at a point removed from the silver-silver chloride electrode, so that an integral reservoir of electrolyte is present between the solution and the electrode.
Potentiometric Dissolved Gas Sensor Prior Art
The carbon dioxide sensor is exemplary of potentiometric gas sensors of the prior-art. U.S. Pat. No. 4,734,184 is one typical example from a large literature of planar carbon dioxide sensors. In this example the device consists of a planar insulating substrate with two conductor elements for connection to a measuring circuit. Assembled thereon are two silver-silver chloride electrodes. One electrode is an internal potentiometric reference electrode, the other electrode is further coated with an integral water permeable layer, then a pH sensing layer constituting together an internal pH indicator electrode. The electrode pair is further coated with two hydrophilic matrixes containing electrolytes, together constituting the integral internal electrolyte, and then a gas permeable membrane. Thus, the potentiometric gas sensor of this typical example requires seven coating steps onto the conductor elements and their insulating support. This device is wet-up prior to use, then immersed in a test solution containing dissolved carbon dioxide. The gas diffuses through the gas permeable membrane into the integral internal electrolyte layer where it dissolves and changes the pH of the electrolyte. The integral internal electrolyte and the two internal electrodes are electrically isolated from the test solution by the gas permeable membrane. The pH change of the internal electrolyte, which is related to the carbon dioxide concentration, is measured by the voltage between the internal indicator and reference electrode.
Simplifications of the classical two-electrode carbon dioxide sensor design have been disclosed in U.S. Pat. No. 5,496,521. This patent describes a carbon dioxide electrode with no internal reference electrode. The device comprises an indicator pH electrode an integral internal electrolyte layer and an ionophore doped homogeneous gas permeable over-layer. The test solution is electrically connected to the integral internal electrolyte by the ion conduction through the homogeneous, ionophore-doped membrane. The sensor of this construction still needs at least four coating layers on the conductor elements and their insulating substrate. Similarly, U.S. Pat. No. 5,554,272 describes a bicarbonate sensor using a homogeneous gas permeable membrane rendered ion conducting by incorporation of an ionophore.
Polarographic Oxygen Sensor Prior-Art
The dissolved oxygen sensor is exemplary of polarographic gas sensors of the prior-art. U.S. Pat. No. 4,534,356 is one typical example from a large literature of planar dissolved oxygen sensors. In this example, the device consists of a planar insulating substrate with two conductor elements for connection to a measuring circuit. There is a coating of silver, then silver chloride on one conductor element that constitutes a first electrode, the reference electrode or anode. A coating of a catalytic metal film (gold or platinum in this example) applied over the other conductor element constitutes the second electrode, the cathode. The electrode pair is further coated with an integral electrolyte layer consisting of a hydrophilic membrane containing dissolved salts and then a second layer which is a gas permeable membrane (Teflon in this example). Thus, this polarographic gas sensor consists of six coating steps for applying the various layers onto the conductor elements and their insulating support. Another typical example is U.S. Pat. No. 5,246,576. In this device there are anode and cathode metal coatings on a planar substrate, with two over-layers. The first is an integral electrolyte layer comprising a hydrophilic membrane containing salts. The second layer is formed from one or two gas permeable membrane coatings. There are a total of eight coating steps in this device. These devices are wet-up prior to use so that the integral electrolyte immersing the electrode pair already contains water and dissolved salts. In use, these devices are immersed into a test solution containing dissolved oxygen. The gas diffuses through the pas permeable membrane and then diffuses through the integral electrolyte to the cathodic electrode surface where it is electrochemically reduced. The internal electrolyte and the two internal electrodes are electrically isolated from the test solution by the gas permeable membrane. The current flowing between the internal anode and cathode is proportional to the oxygen concentration
Modifications to the classical polarographic oxygen sensor design are disclosed in U.S. Pat. No. 5,514,253. This patent describes an oxygen electrode with no internal reference anode. It consists of a cathode coated with an integral electrolyte layer and a gas permeable over-layer. There are openings through the gas permeable over-layer so that the integral electrolyte makes electrical contact with the external test solution well away from the electrode region. This configuration allows the use of an external reference electrode. However, there are still four coating steps required in this example. U.S. Pat. No. 5,078,854 discloses a polarographic oxygen electrode with an integral internal electrolyte and a continuous (homogeneous) gas permeable membrane over-layer. The gas permeable over-layer is rendered appropriately ion conducting by dissolving lipophilic ions into it. As with U.S. Pat. No. 5,514,253, this patent teaches a simplified polarographic electrode with no internal reference electrode. At least three coating steps are required to fabricate this prior-art sensor.
It is thus an essential feature of conventional sensors of the types discussed above that the integral internal electrolyte element is large enough and sufficiently well isolated from the test solution that it behaves as a reservoir which immobilizes the sensor's reagents within it. In these conventional sensors the reservoir's reagent composition thus remains essentially fixed for the duration of a measurement (except in the first few seconds during wet-up of dry stored devices and except for the chemical reaction involving the species to be analyzed whose compositional changes constitute the sensor reaction), and contaminants from the test solution are excluded from and thus at low concentration in this internal electrolyte reservoir. Indeed, it is most often the case that the composition of reagents in the internal electrolyte reservoir element at the electrode surface remains fixed for numerous measurements because these devices have been typically designed to be reusable. In these typical prior-art devices the sensor's internal electrolyte element is completely isolated from the test solution by one or more layers that selectively transport only the species to be analyzed. For example, prior-art dissolved carbon dioxide and oxygen sensors consist of internal electrolyte elements covering the sensors' electrodes and a selectively gas permeable, but electrolyte impermeable over-layer on top of that. In other prior-art devices, where there is direct contact between the internal electrolyte element and the test solution, but the internal electrolyte adjacent the electrode is far removed from the point of contact to the test solution.
For these and other reasons prior-art planar electrochemical sensors have required numerous electrode materials and membrane coatings to achieve the desired functionality. Prior-art planar electrochemical sensors, therefore, are complicated and expensive to produce. In addition, such devices generally still also require at least a single, in-use calibration fluid step to achieve a performance equivalent to laboratory analyzers. Even sensor designs that use micro-fabrication technology (U.S. Pat. Nos. 5,063,081 and 5,514,253 for example) with its high levels of dimensional precision have failed to achieve the standard of performance (reproducible slope and intercept of the response) required for use without a calibration step in a fluidics-free analyzer.
Manufacturers of home use glucose sensors have developed far simpler devices that are manufactured at low cost. Such devices do not require calibration at the point-of-use, but they still require lot-calibrators. However, as is appreciated by those skilled in the art, these devices do not meet the performance requirements of the quantitative laboratory analysis and are classified as semi-quantitative. Thus there remains a significant need to provide electrochemical sensor devices for precise quantitative analysis which are sufficiently simple in design and construction for use as cost-effective unit-use devices.